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Recombinant Zebrafish -Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme

Department of Medical Laboratory Science and Biotechnology, College of Medicine (T.-T.K., W.-N.C., T.-F.F.), and Department of Biochemistry and Molecular Biology, College of Medicine, and Cardiovascular Research Center (H.-L.W., G.-Y.S.), National Cheng Kung University, Tainan, Taiwan

A cDNA encoding for zebrafish -glutamyl hydrolase (GH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-zGH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant zGH was similar to mammalian GH. Thrombin digestion of this NH-zGH fusion protein resulted in zGH with approximately 2-fold higher catalytic activity compared with the NH-zGH fusion enzyme. This recombinant zGH is active and exhibits comparable endopeptidase activity with folate substrate and antifolate drug methotrexate. Use of this recombinant zGH significantly increased efficiency in folylpolyglutamate hydrolysis for folate analysis compared with current protocols.